Method for Freezing and Storing Cells

ABSTRACT

The invention relates to a method for freezing and storing cells in a vessel. Said method comprises the following steps: a) the cells are frozen to a storage temperature; and b) the cells are stored at said storage temperature. The inventive method is characterized in that the cells are stored in a gas-tightly sealed manner and/or at a partial oxygen pressure which is lower than the atmospheric pressure. The invention further relates to frozen cells in freezing medium in a vessel which is gas-tightly sealed and/or in which the partial oxygen pressure is lower than the atmospheric pressure.

The invention is related a method for freezing and storing of cells in acontainer.

In addition, the invention is related to frozen cells in freezing mediumin a container.

The freezing of cells is a method that is often used in laboratories,which work in cell biology (Parkes A. S., Proc. R. Soc. Lond. B. Biol.Sci. 147(929): 424-426, 1957). In order to avoid the detrimental effectsof freezing, which are predominantly caused by the generation of icecrystals, cryoprotective substances such as glycerol or DMSO are usuallyadded to the freezing medium (Pomerat C. M. und P. S. Moorhead, Tex.Rep. Biol. Med. 14(2): 237-253, 1956; Bouroncle B. A., Proc. Soc. Exp.Biol. Med. 119(4): 958-961, 1965; Murthy S. S., Cryobiology 36(2):84-96, 1998). The cells suspended in freezing medium are slowly frozendown with a cooling rate from approximately 1° C. per minute up to −80°C. per minute. The slow cooling down is achieved by individual isolationof the cryotubes, e.g. by a polystyrene box having a suitable wallthickness. The cells frozen in this way can only be preserved for ashort period of approximately 2 to 4 months.

For a long term preservation at lowest temperatures, the transfer of thesamples in liquid nitrogen (−196° C.) or in its gas phase (−150° C. to−160° C.) needs to be done, where in theory an unlimited storage life ofthe frozen cells is possible (Lindl T., Zell-und Gewebekultur, SpektrumAkademischer Verlag GmbH, 4: 110-113, 2000).

Animal und human cells are used in cell-based assays in“high-throughput-screening” (HTS). A continuous culture of the cellsaccording to techniques known in the art is required for this purpose,so that sufficient numbers of cells of constant quality are providedregularly, which can be seeded in the containers used in the assays,usually multiwell plates. However, up to now it is not possible toprepare the cells in the containers, to store them in this form at −80°C. for long terms and to keep them in this way in any amount for theassay. The storage life of cells, which have been frozen at −80° C., islimited to 2 to 4 months. In addition, the storage of multiwell platesand other containers in liquid nitrogen is technically difficult andonly possible with a big requirement for space. Moreover, the need tofreeze the cells down slowly, as described above, makes the processingof a high number of containers more difficult.

The problem underlying the invention is to provide a method, whichallows for the provision of cells for use in cell-based assays in therespective containers in sufficient numbers and constant quality and forlong term storage.

The invention solves this problem by providing a method according toclaim 1 comprising the following steps:

-   -   a) freezing the cells down to a storage temperature,    -   b) storing at said storage temperature,        wherein the storing is performed under gas tight sealing and/or        under reduced oxygen partial pressure as compared to atmospheric        pressure.

First of all, a number of terms are explained, which are used in theinvention.

The term “cell” according to the invention can be a eukaryotic cell thatmeans a cell with a true cell nucleus, as well as a number ofcharacteristic differentiations of the cytoplasm. Such a eukaryotic cellaccording to the invention can be of animal or human origin.

The term “container” used according to the invention can be a “well”(cavity) of a multiwell plate, as well as the entirety of the plate.Likewise, the term “container” can also refer to other containers usedin cell culture, such as e.g. cell culture dishes or cell culturesflasks of a number of sizes, which are suitable for culturing cells.

The term “storage temperature” according to the invention refers to thattemperature, at which the cells are stored, preferably in a deepfreezer. The cells can be stored at this storage temperature for atleast one month up to more than 12 months.

The term “gas tight sealing” according to the invention refers to thesealing of the container, in which the cells are frozen, which iscapable of keeping up an oxygen partial pressure that is reducedcompared to atmospheric pressure, or of keeping protective gas in thecontainer.

The term “atmospheric pressure” according to the invention refers to thetotal air pressure of the surrounding atmosphere consisting of air,which is 1 bar.

The term “oxygen partial pressure” according to the invention refers tothe partial pressure of oxygen in gas form of a total pressure of anatmosphere consisting of air. The oxygen partial pressure in anatmosphere consisting of air, which has a pressure of 1 bar, is 0.21bar.

The term “multiwell plate” according to the invention refers to a plateof a suitable material, such as polystyrene, which comprises a number of“wells” (cavities) for taking up the cells in cell culture medium. Sucha plate can have 6, 12, 24, 48, 96, 384 or 1 536 of such wells.

The term “thermal conductivity” according to the invention has to beunderstood by its known physical definition. A measure for the thermalconductivity of a compound is its thermal conductivity value. Metalshave a high thermal conductivity compared to e.g. glass. The thermalconductance always proceeds from regions of high temperature to regionsof low temperature.

The term “high-throughput-screening” (HTS) refers to assaying aplurality of compounds, cells or suchlike in a relatively short periodof time. For example, the effect of compounds, cells or suchlike oncells in the wells of suitable containers, preferably of a multiwellplate, can be tested in a cell-based assay. Preferably, this can beperformed with a plurality of compounds or cells or suchlike with a highthroughput rate.

The term “freezing medium” comprises solutions, which are suitable forprotecting living cells from cryo-damage during freezing. Such solutionscan comprise culture medium and cryoprotective substances(“cryprotectives”), which can be solvents such as dimethyl sulfoxide(DMSO) or glycerol.

The term “cell-based assay” according to the invention is used for anexperiment (“assay”), in which living cells can be used. The cells canbe used in this assay directly after thawing in the wells of a suitablecontainer. Thus, the cell-based assay can be performed directly in thewells of this container.

The term “vitality” according to the invention refers to the fraction ofliving cells in a container to be tested, e.g. of a multiwell plate, incomparison to cells in a non-frozen container, the “control container”,such as a multiwell plate, as reference. The determination of thefraction of living cells can either be made by staining the cell nucleus(dye exclusion test, Thumm W., Z. Krebsforsch. 60(1): 91-93, 1954), orby testing for metabolic activity (Glass R. H., S. A. Ericsson et al.,Fertil. Steril. 56(4): 743-746, 1991).

The invention has shown that a method as outlined above is very suitablefor providing cells for use in cell-based assays in suitable containersin sufficient numbers and constant quality and for long term storage.

In the following the invention is described further.

Subject of the invention is a method for freezing and storing cells in acontainer comprising the steps of:

-   -   a) freezing the cells down to a storage temperature,    -   b) storing at said storage temperature,        characterized in that the storage is performed under gas tight        sealing and/or under reduced oxygen partial pressure as compared        to atmospheric pressure.

Preferably, cells of a cell culture that is in exponential growth phaseare used for freezing the cells down. Prior to harvesting, preferablysuch an amount of cells is in the container that evenly covers the floorof the container. In the case of a well of a 96-well plate thispreferably equates to 80 to 200 μl, more preferably 85 to 190 μl, mostpreferably 93 to 188 μl of culture medium with cells per cm² surface.

After harvesting the cells can be re-suspended in freezing medium at acell density of preferably 0.05-10×10⁶ cells, more preferably at0.1-5×10⁶ cells per ml. The freezing medium can comprise culture medium,as well as a cryoprotective. The cryoprotective can be glycerol ordimethyl sulfoxide (DMSO). The final concentration of the cryoprotectivecan be 1 to 20% per vol., preferably 2 to 15% per vol., most preferably4 to 10% per vol. Examples for suitable freezing media are a mixture of95% DMEM (Dulbecco's Modified Eagels Medium) complete medium, 5%glycerol, as well as of 95% DMEM complete medium, 5% DMSO, whereincomplete medium can consists of DMEM, 4 mM L-Glutamine, 2 mM sodiumpyruvate and 10% fetal calf serum (FCS).

The culture medium used in the freezing medium can be a different mediumthan DMEM. Every other medium used in cell culture can be used, such asRPMI or neurobasal medium. A salt solution in place of the cell culturemedium can also be used in the freezing medium, such as HBSS (Hank'sBalanced Salt Solution) or other common salt solutions.

The cells resuspended in freezing medium can at the most be frozen 60min, preferably at the most 45 min, most preferably at the most 30 minafter resuspending.

The reduction of the oxygen partial pressure can be performed byreducing the pressure and/or by gassing with a protective gas.

The reduction of the oxygen partial pressure can preferably be doneprior to freezing.

The oxygen partial pressure in the container can be reduced to 52.5 mbarto 0.21 mbar, preferably to 26 mbar to 0.21 mbar, most preferably to 13mbar to 0.21 mbar.

The gas tight sealing of the container according to the invention can bemade by shrink-wrapping the container in a gas tight film tube or byapplying a gas tight adhesive film. In doing this, a suitable containercan be closed with a lid and shrink-wrapped under vacuum in a gas tightfilm tube that is preferably made from polyethylene. The container canbe carefully moved in the tube, which is then closed at one side. Usinga commercially available shrink-wrapping device, the remaining airoxygen can be removed from the film tube and the film can beshrink-wrapped. Such a shrink-wrapping device can be, e.g. a Foliovacuum-shrink-wrapping device. For comparison, suitable containers canbe frozen under the same conditions, however under an oxygen partialpressure that is reduced compared to the atmospheric pressure.

After gas tight sealing of the container a residual volume of airreferring to atmospheric pressure can remain in the container, that ispreferably up to 17.5 times, more preferably up to 15 times, morepreferably up to 7 times, more preferably equal to the volume of thecell suspension in the container.

For example, such a container can be a multiwell plate, preferably a96-well plate. A residual volume of air in comparison to the atmosphericpressure can remain in the 96-well plate with a volume of 25 μl cellsuspension per well under gas tight sealing, which can be mostpreferably up to 17.5 times the volume of the cell suspension.

The protective gas according to the invention can be selected from thegroup consisting of nitrogen, helium and argon.

The cells, which are frozen with the method according to the invention,can be eukaryotic cells. The cells that are frozen with the methodaccording to the invention can be of animal or human origin. Forexample, these cells can be murine cells, hamster cells, monkey cells orpig cells. The cells, which are frozen with this method can further beestablished cell lines, as well as primary or secondary cells. Thecells, which are frozen with the method according to the invention, canfurther be adherent cells, or cells that grow in suspension. The cells,which are frozen with this method, can comprise a plurality of celltypes. These cell types can be selected from the group consisting ofcells of ectodermal origin, mesodermal origin, endodermal origin andgerm cells. Furthermore, the cell types can be selected from the groupconsisting of fibroblasts, epithelial cells, haematopoietic cells andneural cells. For example, the cells can be L-292 mouse fibroblasts,HeLa-cells or HCT-116 cells.

As contemplated in the invention, the cells can be frozen down to thestorage temperature with a cooling rate, which can be at least 20° C.per minute, preferably at least 40° C. per minute, more preferably atleast 64.5° C. per minute, more preferably at least 80° C. per minute.The cooling rate according to the invention, as compared to techniquesknown in the art, allows for a rapid cooling, and even for a coolingbelow the freezing point of the cell suspension present in thecontainer. Together with the medium it is possible to suppress theformation of crystals in the system and to keep the solution in thatglass-like state, which is necessary for storing the cells at lowesttemperatures without causing damage. A cooling rate according to theinvention can most preferably be 64.5° C. per minute.

The storage temperature according to the invention can be not lower than−85° C., more preferably not lower than −65° C., more preferably notlower than −60° C., more preferably not lower than −50° C. According tothe invention the storage temperature can be maintained in a deepfreezer. As contemplated by the invention, a particularly preferredstorage temperature can be not lower than −65° C.

The freezing can be achieved by contact of at least one surface of thecontainer with a material of suitable (high) thermal conductivity, whichhas been pre-cooled on a suitable temperature. The material of suitablethermal conductivity can be a metal. The suitable temperature can bebetween −90° C. and −50° C., preferably between −85° C. and −65° C.

The storage of the cells according to the invention can be performedwithout the use of liquid nitrogen. Preferably, the storage of the cellsis done in a deep freezer.

The container can be selected from the group consisting of multiwellplate, cell culture dish and cell culture flask.

The multiwell plate can be selected from the group consisting of 6-well,12-well, 24-well, 48-well, 96-well, 384-well and 1 536-well plate.

The invention further relates to frozen cells in freezing medium in acontainer under gas tight sealing and/or under oxygen partial pressurethat has been reduced in comparison to the atmospheric pressure.

The oxygen partial pressure in the container can be 52.5 mbar to 0.21mbar, preferably 26 mbar to 0.21 mbar, most preferably 13 mbar to 0.21mbar.

The cells according to the invention can be used directly after thawingin freezing medium in the container in a cell-based assay withoutpassaging. This can be performed without washing, culturing or passagingthe cells to a different culture dish. Approximately 1 to 8 hours afterthawing at 37° C., culture medium can be added to the wells in thecontainer, preferably in a suitably tempered cell incubator withsuitable CO₂-atmosphere. In doing so, the freezing medium surroundingthe cells is diluted. Preferably, this can be a 1:1 dilution. After theaddition of culture medium, the cells can be used directly in thecontainer for a cell-based assay. This can be done without performingwashing, culturing or passaging of the cells to a different container.In the following cell-based assay, controls can be done with and withoutthe solvent that has been used as cryoprotective in the respectivepercentage, in order to test for a potential effect of the solvent onthe assay.

The cell-based assay can be performed in the form of ahigh-throughput-screening (HTS). The effect of e.g. compounds, cells orsuchlike can be tested on cells in wells of a container, preferably amultiwell plate. Preferably, this is performed with a plurality ofcompounds or cells with a preferably high throughput rate. The testedcompounds can preferably be pharmacologically active compounds. Thetested compounds can, in addition, be preferably ligands for cellularreceptors. The tested compounds can particularly preferably have aneffect of the gene expression in the cells of the wells. The testedcells can have an effect on the cells in the wells. One or morecompounds or cells per well can be tested.

The frozen cells can have a vitality at a storage temperature lower thanor equal to −65° C. for at least one month, preferably more than 2months, more preferably more than 4 months, most preferably more than 12months of above 70%, preferably above 80%, most preferably of above 95%of the vitality of cells of a non-frozen control container. In the caseof large containers from which the cells can easily be removedcompletely, the vitality of cells can be determined by the dye exclusionassay that is known in the art (Thumm W., Z. Krebsforsch. 60(1): 91-93,1954). For smaller containers, from which the frozen cells can not betaken reliably for determining the vitality, the vitality assay can beperformed indirectly via the determination of the metabolic activity ofthe cells. A substrate, e.g. resazurin, is added to the cells and isreduced by metabolically active cells. The absorption spectrum orfluorescence spectrum of the substrate is changed by the reduction,which can easily be quantified (Glass R. H., S. A. Ericsson et al.,Fertil. Steril. 56(4): 743-746, 1991). After preceding grading of themeasured absorption or fluorescence by using cell suspensions with aknown cell count, quantifying of the vitality can be performed. Themeasurement of the metabolic activity of the cells can, apart fromresazurin, be performed with other dyes known in the state of the art,such as MTT, XTT or neutral red.

The invention is further explained in the following examples.

EXAMPLE 1

Materials and devices:

-   1. L-929 murine fibroblast cell line of ATCC (American Type Culture    Collection), No.: CCL-1-   2. DMEM culture medium (Dulbecco's Modified Eagels Medium)-   3. L-glutamine-   4. sodium pyruvate-   5. fetal calf serum (FCS)-   6. glycerol-   7. PBS (Phosphate Buffered Saline) Trypsin/EDTA    (Ethylene-diamine-tetraacetic acid) 0.5 g/l und 0.25 g/l-   8. resazurin-   9. 96-well plates-   10. polyethylene tube film-   11. Folio vacuum shrink-wrapping device-   12. sterile working bench-   13. CO₂ cell culture incubator-   14. −80° C. deep freezer

L-292 mouse fibroblasts were cultured according to the standard protocolof the ATCC in DMEM complete medium (DMEM, 4 mM L-glutamine, 2 mM sodiumpyruvate, 10% FCS) until a sufficient number of cells was provided forfreezing of the cells. For harvesting, the surfaces of the containersshould be approximately 80 to 90% confluently grown with cells. Theculture supernatant was decanted and the layer of cells was washed oncewith PBS. 1 ml trypsin/EDTA were applied on the cell layer per 25 cm²surface, equally dispersed and incubated at room temperature (RT) for 5minutes (min). The number and vitality of the detached cells wasdetermined, which were then pelleted at 180×g for 4 min at RT. Thesupernatant was discarded and the cell pellet was re-suspended at a celldensity of 6×10⁵ cells/ml in freezing medium (95% DMEM complete medium,5% glycerol). 50 μl of the cell suspension were pipetted in each well ofa 96-well plate, wherein special care was taken that the cell suspensionwas covering the surface of the wells. The 96-well plates were closedwith the lid and each of them shrink-wrapped under vacuum in a film tubemade from polyethylene. The plates were carefully placed in the filmtube, which was then sealed at one side. Using a commercially availableshrink-wrapping device the remaining air was removed from the film tubeas far as possible and the film was shrink-wrapped. For comparison,96-well plates were frozen under the same conditions, however withoutremoving the air. The shrink-wrapped multiwell plates were placeddirectly on the metal floors of a −80° C. deep freezer, and frozen andstored in there. The plates were not stacked, and the entire freezingprocess was done rapidly. The time period from adding the freezingmedium to starting the freezing process was no more than 45 min in thisexample.

The cells could be stored in 96-well plates under such conditions formore than 1.5 years and thawed again with a vitality of more than 90%.However, when cells were not frozen under vacuum, their storage life waslimited to 2 to 4 months. The determination of the vitality was donedirectly in the 96-well plate using the reduction of resazurin (Glass R.H., S. A. Ericsson et al., Fertil. Steril. 56(4): 743-746, 1991).

The cells could be stored under such conditions for more than 12 monthsand could be thawed in the 96-well plates with a vitality of more than90% of a none-frozen plate. In contrast, the vitality of the controlplate, which had not been treated with the method was only just above20%.

FIG. 1 shows the result of the experiment of Example 1.

EXAMPLE 2

The cell line HCT-116 was frozen using the method according to theinvention. For comparison, HCT-116 cells were frozen in freezing mediumwith DMSO (95% DMEM complete medium, 5% DMSO) and with glycerol (95%DMEM complete medium, 5% glycerol) in 96-well plates. The frozen cellswere thawed again after 12 months and the cell vitality was determinedby reduction of the dye resazurin. The cell activity is given asrelative signal intensity in [U].

For analyzing a HTS-assay the Z-factor is used in the state of the art.The Z-factor is a statistical value, which is calculated from theaverage values and the standard deviations of the measured values. Anideal assay has a Z-factor, which is around 1. Assays are preformed wellbetween 1 and 0.5. Is the Z-factor falling below 0.5, the separatingarea between control and sample is getting too small, so that a cleardistinction of the cell activity is not possible anymore.

FIG. 2 shows graphically the result of Example 2.

FIG. 3 shows the raw data of Example 2.

EXAMPLE 3

In order to show the functionality of cells frozen according to theinvention, cells were used in a cytotoxicity assay after thawing.HeLa-cells were frozen in 96-well plates using the method according tothe invention, then thawed after 13 months and their IC₅₀-value ofNa₂SeO₃ determined. To do this, 12 dilutions of Na₂SeO₃ were added tothe cells. The dilutions were selected such, that the concentration ofNa₂SeO₃, which kills 50% of the cells, was in the middle of the testedconcentration range. The depicted values are average values of aquadruplicate. After incubating the test substance for 48 hours at cellculture conditions (37° C., 5% CO₂ and high air humidity), the cellcount of living cells was determined by testing for the metabolicactivity of living cells. For this purpose, resazurin was used again,which is reduced in the mitochondria of metabolically active, livingcells, and which turns into the fluorescent resofurin. The IC₅₀-value isgiven by that concentration, at which only 50% of the cell activity canbe detected.

FIG. 4 shows the result of Example 3.

The examples show that the method according to the invention isparticularly suitable for providing cells for use in cell-based assaysin the respective containers in sufficient numbers and constant qualityand for storing them for long terms.

1-21. (canceled)
 22. A method of freezing and storing cells in acontainer comprising the steps of: a) freezing a cell suspensioncomprising said cells down to a storage temperature; b) storing saidcell suspension at said storage temperature; wherein the storing isperformed under gas tight sealing and/or under reduced oxygen partialpressure as compared to atmospheric pressure.
 23. The method of claim 1,wherein the reduction of the oxygen partial pressure is done by reducingthe pressure and/or by gassing with a protective gas.
 24. The method ofclaim 1, wherein the reduction of the oxygen partial pressure is doneprior to freezing.
 25. The method of claim 1, wherein the oxygen partialpressure in the container is reduced to between about 52.5 mbar and 0.21mbar.
 26. The method of claim 25, wherein the oxygen partial pressure inthe container is reduced to between about 26 mbar to 0.21 mbar.
 27. Themethod of claim 26, wherein the oxygen partial pressure in the containeris reduced to between about 13 mbar to 0.21 mbar.
 28. The method ofclaim 1, wherein the gas tight sealing of the container is made byshrink-wrapping in a gas tight film tube or by applying a gas tightadhesive film.
 29. The method of claim 1, wherein a residual volume ofair remains in the container after gas tight sealing of the container,wherein said residual volume is up to about 17.5 times the volume of thecell suspension in the container.
 30. The method of claim 29, whereinsaid residual volume of air is up to about 15 times the volume of thecell suspension in the container.
 31. The method of claim 30, whereinsaid residual volume of air is up to about 7 times the volume of thecell suspension in the container.
 32. The method of claim 31, whereinsaid residual volume of air is equal to the volume of the cellsuspension in the container.
 33. The method of claim 2, wherein theprotective gas is selected from a group consisting of nitrogen, helium,and argon.
 34. The method of claim 1, wherein the cells are of animal orof human origin.
 35. The method of claim 1, wherein said freezing ofsaid cell suspension down to said storage temperature is with a coolingrate of at least 20° C. per minute.
 36. The method of claim 35, whereinsaid freezing of said cell suspension down to said storage temperatureis with a cooling rate of at least 40° C. per minute.
 37. The method ofclaim 36, wherein said freezing of said cell suspension down to saidstorage temperature is with a cooling rate of at least 64.5° C. perminute.
 38. The method of claim 37, wherein said freezing of said cellsuspension down to said storage temperature is with a cooling rate of atleast 80° C. per minute.
 39. The method of claim 1, characterized inthat the storage temperature is not lower than about −85° C.
 40. Themethod of claim 39, characterized in that the storage temperature is notlower than about −65° C.
 41. The method of claim 40, characterized inthat the storage temperature is not lower than about −60° C.
 42. Themethod of claim 41, characterized in that the storage temperature is notlower than about −50° C.
 43. The method of claim 1, wherein saidfreezing is achieved by contact of at least one surface of the containerwith a material of suitable thermal conductivity that has beenpre-cooled to a suitable temperature.
 44. The method of claim 43,wherein said material is not metal.
 45. The method of claim 43, whereinsaid material is pre-cooled to a temperature between about −90° C. and−50° C.
 46. The method of claim 43, wherein said material is pre-cooledto a temperature between about −85° C. and −65° C.
 47. The method ofclaim 1, wherein said storing of said cells is performed without the useof liquid nitrogen.
 48. The method of claim 1, wherein the container isselected from the group consisting of 6-well, 12-well, 24-well, 48-well,96-well, 384-well and 1,536-well plates.
 49. A composition comprisingfrozen cells in freezing medium in a container under gas tight sealingand/or under an oxygen partial pressure that has been reduced incomparison to the atmospheric pressure.
 50. The composition of claim 49,wherein the oxygen partial pressure in the container is between about52.5 mbar and 0.21 mbar.
 51. The composition of claim 49, wherein theoxygen partial pressure in the container is between about 26 mbar to0.21 mbar.
 52. The composition of claim 49, wherein the oxygen partialpressure in the container is between about 13 mbar to 0.21 mbar.
 53. Thecomposition of claim 49, wherein the cells can be used directly afterthawing in freezing medium in the container in a cell-based assaywithout passaging.
 54. The composition of claim 53, wherein thecell-based assay is done in the form of a high-throughput-screening(HTS).
 55. The composition of claim 49, wherein after storage at astorage temperature lower than or equal to about −65° C. for at leastone month, said frozen cells have a vitality of above 70% of thevitality of cells of a non-frozen control container.
 56. The compositionof claim 55, wherein said frozen cells have a vitality of above 80% ofthe vitality of cells of a non-frozen control container.
 57. Thecomposition of claim 56, wherein said frozen cells have a vitality ofabove 90% of the vitality of cells of a non-frozen control container.58. The composition of claim 55, wherein said storage at said storagetemperature is for at least 2 months.
 59. The composition of claim 55,wherein said storage at said storage temperature is for at least 4months.
 60. The composition of claim 55, wherein said storage at saidstorage temperature is for at least 12 months.